Tumor necrosis factor-? (TNF), originally described as a death-inducer in tumor cells, is also known as an effector molecule of NK-cells and cytotoxic T-lymphocytes (CTL) involved in tumor rejection. However, accumulating evidence suggests that TNF can also be a tumor promoting factor. We observed expression of membrane-associated TNF on the surface of neuroblastoma (NB) cells derived from humans or mice. Disruption of TNF:TNF-R(s) interactions, using blocking antibodies or soluble TNF-R2, decreased human NB cell viability indicating that TNF constitutively generates a pro- survival signal. Similar data were obtained using NB tumors spontaneously arising in transgenic TH- MYCN mice. Neutralization of TNF was also associated with downregulation of MHC class I in NB cells and led to their decreased susceptibility to perforin and granzyme B, the major components of cytotoxic granules of CTLs and NK cells. We also observed loss of tumor cell capacity to trigger the inhibitory CD94 heterodimer on cytotoxic lymphocytes. Thus, TNF appears to induce phenotypic changes with potentially opposing effects on CTL and NK recognition of NB cells. However, our results suggest that decreased recognition of NB-cell by both CTLs and NK cells is the net effect of autochthonous TNF activity. This research program proposes to use primary human and mouse NB cells as a model for dissecting the role of tumor-produced TNF in regulating survival, antigen presentation and sensitivity to perforin/granzyme mediated death in tumor cells. The in vivo effects of TNF blockade in NB cells will be investigated using spontaneous model of murine neuroblastoma and xenotransplantation of human NB cells into the immunodeficient mice. We will also characterize TNF- induced signals and pathways regulating susceptibility to cytotoxic lymphocytes either at the level of target sensitivity or effector activation. Detailed knowledge of such signals will improve our understanding of the pathogenesis of autoimmune disorders, allograft rejection and tumor immune escape. We believe that our research will also shed light on the molecular mechanisms of NB tumorogenesis and lead to the development of new strategies of NB therapy based on blockade of endogenous TNF. Importantly, a spectrum of TNF blockers is already approved and successfully applied in clinical settings. Characterization of TNF function in neuroblastoma will prompt investigations on the role of this cytokine in the pathogenesis of other tumors.

Public Health Relevance

We observed that membrane bound Tumor necrosis factor (TNF) alfa is expressed on the surface of neuroblastoma (NB) cells and its functional blockade leads to decreased tumor viability and increased tumor susceptibility to killing by cytotoxic lymphocytes. Investigations on the role of tumor-produced TNF in regulating survival, proliferation and antigenic characteristics of NB cells will improve our understanding of the pathogenesis of autoimmune disorders, allograft rejection and tumor immune escape.